The critical events that occur during early pregnancy include dramatic growth and development of the embryo/fetus and also placentation, which includes vascularization of the placenta. In fact, the majority of embryonic loss, amounting to 30 to 50% of fertilized oocytes, occurs during early pregnancy in all mammals, including humans. Steroids 'drive' blood flow to the pregnant uterus, placental angiogenesis and angiogenic factor expression. Recently, we have shown dramatic increase in endometrial angiogenesis during early pregnancy and that day of early pregnancy as well as assisted reproductive techniques (ART) affect not only endometrial angiogenesis but also expression and localization of estrogen and progesterone receptors (ER and PR) in the early utero-placenta. Thus, to better understand their physiological role, we need to quantify expression and evaluate localization of ER and PR in the utero-placental vasculature and other tissue compartments in pregnancies after natural mating and also after ART. We also must evaluate the functional consequences of altered utero-placental steroid receptor activation in vitro and in vivo. Therefore, the Long- Term Goal of this project is to determine the role of estrogens and progesterone and their receptors in the regulation of utero-placental growth and development, and especially vascularization, during the critical early stages of normal and compromised pregnancies. To meet this goal, we will evaluate placental expression of nuclear and membrane ER and PR after natural mating and after transfer of embryos produced in vivo or in vitro. Our overall hypothesis is that expression and/or function of ER and PR changes during early pregnancy and, in addition, is compromised during early pregnancy in utero-placental tissues after application of ART. We will evaluate expression of ER and PR protein using immunofluorescence (IF) followed by image analysis as well as Western blot, and mRNA expression using laser capture micro-dissection followed by qPCR. The utero-placental tissues we will use were already collected from other recent studies and comprise tissues obtained during early pregnancy after natural mating (Animal Model 1, Aim 1) and after transfer of embryos produced in vivo or in vitro (Animal Model 2, Aim 2). Our specific hypotheses are that expression and localization of ER and PR is differentially regulated as normal pregnancy progresses (Aim 1, hypothesis 1), and is altered by application of ART (Aim 2, hypothesis 2). Lastly, for these studies we will use our well- characterized domestic animal models of compromised pregnancy that have previously been shown to have relevance not only to agriculture but also to human health. Because of the importance of sex steroids in regulating utero-placental vascular function and development during early pregnancy, the results of these studies will have major implications for pregnancy establishment and will lead to new and informed strategies and therapeutic tools to improve pregnancy success and prevent spontaneous abortion, implantation failure and/or other disorders, which has enormous implications for maternal and fetal health and well-being.